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LOX expression and functional analysis in astrocytomas and impact of IDH1 mutation.

da Silva R, Uno M, Marie SK, Oba-Shinjo SM - PLoS ONE (2015)

Bottom Line: The results demonstrate that increased expression and activity of LOX, BMP1 and HIF1A were positively correlated with the malignant grade of astrocytomas.Glioblastoma with a mutation in IDH1 expressed lower levels of LOX in the nucleus, and IDH1-mutated cases showed lower LOX expression levels when compared to wild-type IDH1 cases.Taken together, these results corroborate the role of LOX in the migration, invasion and angiogenesis of astrocytomas.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cellular Biology, Department of Neurology, Faculdade de Medicina da Universidade de São Paulo, São Paulo, 01246-903, Brazil.

ABSTRACT
Lysyl oxidase (LOX) is involved in vital biological processes such as cell motility, cell signaling and gene regulation. Deregulation of this protein can contribute to tumor formation and progression. Although it is known that LOX is involved in invasion, proliferation and tumor migration in other types of tumors, studies of LOX in astrocytomas of different grades are scarce. The purpose of our study was to characterize LOX, BMP1 and HIF1A expression by real-time PCR in astrocytomas with WHO grades I to IV compared to non-neoplastic brain tissue. IDH1 mutational status was determined by PCR and sequencing. LOX protein expression was also analyzed by immunohistochemistry. LOX functional analyses were performed using siRNA knockdown and the specific inhibitor BAPN in two glioblastoma cell lines. The expression levels of LOX, BMP1 and HIF1A were correlated and analyzed according to IDH1 mutation status and to the clinical end-point of overall survival of glioblastoma patients. The results demonstrate that increased expression and activity of LOX, BMP1 and HIF1A were positively correlated with the malignant grade of astrocytomas. LOX protein expression also increased according to the degree of malignancy, with localization in the cytoplasm and nucleus and staining observed in endothelial cells. Glioblastoma with a mutation in IDH1 expressed lower levels of LOX in the nucleus, and IDH1-mutated cases showed lower LOX expression levels when compared to wild-type IDH1 cases. LOX knockdown and inhibition by BAPN in U87MG and A172 cell lines affected migration, invasion and soft agar colony formation. Taken together, these results corroborate the role of LOX in the migration, invasion and angiogenesis of astrocytomas. Furthermore, LOX expression is influenced by IDH1 mutational status. This work provides new insights for researchers aiming to design targeted therapies to control astrocytomas.

No MeSH data available.


Related in: MedlinePlus

Effect of LOX knockdown and inhibition in the migratory phenotype of GBM cell lines.LOX expression in U87MG and A172 cell lines transfected with LOX siRNA relative to that of cells transfected with the non-targeted control (NTC) siRNA was evaluated 2 days after transfection (A). The data show the average of two independent experiments, and the vertical bar represents the standard deviation. Western blots of LOX protein were analyzed after transfection with siRNA targeted against LOX and non-targeted control (NTC) siRNA (B). Evaluation of the migratory behavior of U87MG (C) and A172 (D) cell lines with no treatment (parental), transfected with NTC siRNA and siRNA specific for LOX and treated with the LOX inhibitor BAPN. A total of 2.5 x 104 cells were seeded in the upper chamber of Transwell inserts. Cells in the lower chamber were fixed and stained after 18 hours of incubation. Graphs represent the number of migrated cells per field in each condition (means ± standard errors of the means) in two independent experiments. The pictures show representative fields of cells that crossed the insert in all conditions stained with crystal violet at 40x magnification (Mann-Whitney test, **p<0.001; ***p<0.0001).
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pone.0119781.g003: Effect of LOX knockdown and inhibition in the migratory phenotype of GBM cell lines.LOX expression in U87MG and A172 cell lines transfected with LOX siRNA relative to that of cells transfected with the non-targeted control (NTC) siRNA was evaluated 2 days after transfection (A). The data show the average of two independent experiments, and the vertical bar represents the standard deviation. Western blots of LOX protein were analyzed after transfection with siRNA targeted against LOX and non-targeted control (NTC) siRNA (B). Evaluation of the migratory behavior of U87MG (C) and A172 (D) cell lines with no treatment (parental), transfected with NTC siRNA and siRNA specific for LOX and treated with the LOX inhibitor BAPN. A total of 2.5 x 104 cells were seeded in the upper chamber of Transwell inserts. Cells in the lower chamber were fixed and stained after 18 hours of incubation. Graphs represent the number of migrated cells per field in each condition (means ± standard errors of the means) in two independent experiments. The pictures show representative fields of cells that crossed the insert in all conditions stained with crystal violet at 40x magnification (Mann-Whitney test, **p<0.001; ***p<0.0001).

Mentions: LOX knockdown was evaluated after 2 days of transfection of U87MG and A172 GBM cell lines with siRNA targeted against LOX or non-targeted control (NTC) siRNA. The efficiency of transfection was analyzed by RT-qPCR (Fig. 3A) and western blotting (Fig. 3B) for each of the duplicate experiments. Both cell lines presented an approximate 80% decrease in LOX mRNA expression when compared to NTC. Protein expression was also confirmed to be diminished after LOX knockdown with siRNA.


LOX expression and functional analysis in astrocytomas and impact of IDH1 mutation.

da Silva R, Uno M, Marie SK, Oba-Shinjo SM - PLoS ONE (2015)

Effect of LOX knockdown and inhibition in the migratory phenotype of GBM cell lines.LOX expression in U87MG and A172 cell lines transfected with LOX siRNA relative to that of cells transfected with the non-targeted control (NTC) siRNA was evaluated 2 days after transfection (A). The data show the average of two independent experiments, and the vertical bar represents the standard deviation. Western blots of LOX protein were analyzed after transfection with siRNA targeted against LOX and non-targeted control (NTC) siRNA (B). Evaluation of the migratory behavior of U87MG (C) and A172 (D) cell lines with no treatment (parental), transfected with NTC siRNA and siRNA specific for LOX and treated with the LOX inhibitor BAPN. A total of 2.5 x 104 cells were seeded in the upper chamber of Transwell inserts. Cells in the lower chamber were fixed and stained after 18 hours of incubation. Graphs represent the number of migrated cells per field in each condition (means ± standard errors of the means) in two independent experiments. The pictures show representative fields of cells that crossed the insert in all conditions stained with crystal violet at 40x magnification (Mann-Whitney test, **p<0.001; ***p<0.0001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4366168&req=5

pone.0119781.g003: Effect of LOX knockdown and inhibition in the migratory phenotype of GBM cell lines.LOX expression in U87MG and A172 cell lines transfected with LOX siRNA relative to that of cells transfected with the non-targeted control (NTC) siRNA was evaluated 2 days after transfection (A). The data show the average of two independent experiments, and the vertical bar represents the standard deviation. Western blots of LOX protein were analyzed after transfection with siRNA targeted against LOX and non-targeted control (NTC) siRNA (B). Evaluation of the migratory behavior of U87MG (C) and A172 (D) cell lines with no treatment (parental), transfected with NTC siRNA and siRNA specific for LOX and treated with the LOX inhibitor BAPN. A total of 2.5 x 104 cells were seeded in the upper chamber of Transwell inserts. Cells in the lower chamber were fixed and stained after 18 hours of incubation. Graphs represent the number of migrated cells per field in each condition (means ± standard errors of the means) in two independent experiments. The pictures show representative fields of cells that crossed the insert in all conditions stained with crystal violet at 40x magnification (Mann-Whitney test, **p<0.001; ***p<0.0001).
Mentions: LOX knockdown was evaluated after 2 days of transfection of U87MG and A172 GBM cell lines with siRNA targeted against LOX or non-targeted control (NTC) siRNA. The efficiency of transfection was analyzed by RT-qPCR (Fig. 3A) and western blotting (Fig. 3B) for each of the duplicate experiments. Both cell lines presented an approximate 80% decrease in LOX mRNA expression when compared to NTC. Protein expression was also confirmed to be diminished after LOX knockdown with siRNA.

Bottom Line: The results demonstrate that increased expression and activity of LOX, BMP1 and HIF1A were positively correlated with the malignant grade of astrocytomas.Glioblastoma with a mutation in IDH1 expressed lower levels of LOX in the nucleus, and IDH1-mutated cases showed lower LOX expression levels when compared to wild-type IDH1 cases.Taken together, these results corroborate the role of LOX in the migration, invasion and angiogenesis of astrocytomas.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cellular Biology, Department of Neurology, Faculdade de Medicina da Universidade de São Paulo, São Paulo, 01246-903, Brazil.

ABSTRACT
Lysyl oxidase (LOX) is involved in vital biological processes such as cell motility, cell signaling and gene regulation. Deregulation of this protein can contribute to tumor formation and progression. Although it is known that LOX is involved in invasion, proliferation and tumor migration in other types of tumors, studies of LOX in astrocytomas of different grades are scarce. The purpose of our study was to characterize LOX, BMP1 and HIF1A expression by real-time PCR in astrocytomas with WHO grades I to IV compared to non-neoplastic brain tissue. IDH1 mutational status was determined by PCR and sequencing. LOX protein expression was also analyzed by immunohistochemistry. LOX functional analyses were performed using siRNA knockdown and the specific inhibitor BAPN in two glioblastoma cell lines. The expression levels of LOX, BMP1 and HIF1A were correlated and analyzed according to IDH1 mutation status and to the clinical end-point of overall survival of glioblastoma patients. The results demonstrate that increased expression and activity of LOX, BMP1 and HIF1A were positively correlated with the malignant grade of astrocytomas. LOX protein expression also increased according to the degree of malignancy, with localization in the cytoplasm and nucleus and staining observed in endothelial cells. Glioblastoma with a mutation in IDH1 expressed lower levels of LOX in the nucleus, and IDH1-mutated cases showed lower LOX expression levels when compared to wild-type IDH1 cases. LOX knockdown and inhibition by BAPN in U87MG and A172 cell lines affected migration, invasion and soft agar colony formation. Taken together, these results corroborate the role of LOX in the migration, invasion and angiogenesis of astrocytomas. Furthermore, LOX expression is influenced by IDH1 mutational status. This work provides new insights for researchers aiming to design targeted therapies to control astrocytomas.

No MeSH data available.


Related in: MedlinePlus